The Role of the CNOT3 Subunit of the CCR4-NOT Complex in Cellular Senescence

Abstract

Okinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyCellular senescence is a fundamentally irreversible cell cycle arrest associated with upregulated inflammatory responses. Cellular senescence is involved in numerous biological processes, including tumor suppression and organismal aging. Uncovering its mechanism will contribute to the development of novel therapeutic strategies for age-associated diseases as well as cancer. However, the molecular regulators involved in senescence are not fully elucidated. The CCR4- NOT complex, known for its role in gene expression and mRNA decay, is one such regulator. Despite its broad functions, the specific function of the CCR4-NOT complex, particularly the CNOT3 subunit, in cellular senescence remains unknown. In this thesis work, I studied the role of the CCR4-NOT deadenylase complex in cellular senescence, focusing on the regulatory interplay between the CNOT3 subunit and long non-coding RNA (lncRNA). LncRNAs were chosen for their emerging role in regulating gene expression and senescence. Here, I show that the downregulation of CNOT3 is sufficient for cellular senescence induction in A549 human non-small cell lung cancer cells. Knockdown of CNOT3 causes the upregulation of multiple cellular senescence hallmarks, such as CDKN1A, TP53, the senescence-associated secretory phenotype (SASP) (IL6, IL8, and STC1), and senescence-associated β-galactosidase activity. Moreover, these senescence hallmarks are more significantly upregulated when CNOT3 downregulation is combined with PLK-1 inhibitor (BI2536), which reduces the dividing cell population. These results suggest that CNOT3 downregulation could enhance the susceptibility of lung cancer cells to chemotherapy. I also explored the regulatory network involving CNOT3 and lncRNA. Previously, it has been reported that lncRNA can interact with RNA and RNAbinding proteins to regulate gene expression at the post-transcriptional level by determining the fate of target molecules. To investigate the relationship between CNOT3 and lncRNAs, I analyzed microarray RNA-seq data from CNOT3 knockdown in A549 cells and identified a subset of lncRNAs that were upregulated upon CNOT3 depletion. Among them, the lncRNA MAGI2-AS3 was found to influence the cellular senescence pathway, suggesting that CNOT3 and lncRNAs collaboratively regulate cellular senescence. This thesis highlights the multilayered regulatory roles of CNOT3 and its lncRNA partners, offering a novel perspective on enhancing chemotherapy efficacy.doctoral thesi